Proteases (or proteinases) are a subclass of hydrolases (one of the six classes of enzymes designated by the International Union of Biochemistry). The proteases form a highly complex group of enzymes which vary enormously in their physical, chemical and catalytic properties. However, all hydrolyze the peptide bonds between amino acids comprising the structural units of proteins. Thus, the proteolytic enzymes, effective intra- or extracellularly, play an important role in the metabolic and regulatory processes of animal and plant cells, as well as in prokaryotic and eucaryotic microorganisms.
Until about 15 years ago proteases were regarded as degradative enzymes which could only catalyze the total hydrolysis of proteins. However, recent advances in assay techniques, such as the use of more selective substrates, have demonstrated that proteolytic enzymes carry out highly specific and selective modifications of proteins by limited hydrolysis. Extracellular proteases (or exoproteases) are involved primarily in protein turnover, while intracellular enzymes also play a key role in the regulation of metabolic processes and the balance between protein synthesis and degradation.
Protein turnover eliminates abnormal proteins and is essential for the adaptation of cells to new environmental conditions. It is a continual process in all living cells. The cell utilizes proteases to break down polypeptides into their primary components when those polypeptides have no further value to the cell. Thus, a pool of amino acids are provided as precursors for the synthesis of essential proteins and a nitrogen source for nucleotide biosynthesis. The process is similar in all organisms, with different rates of turnover for individual proteins and subcellular fractions.
A number of mechanisms operate to control proteolysis. These include, for example, modulation of substrate proteins by covalent interconversion, change in hydrophobicity and interaction with various molecules, thereby affecting the susceptibility of the molecules to proteolysis. Protease activity is also controlled by nutritional conditions and catabolite repression. For example, in both microbial and mammalian tissues, protease activity has been shown to increase when the organism is placed under conditions of nutrient starvation. Thus, when the cell is starved, proteins serving newly required functions in the cell can be synthesized with little net change in protein content. In contrast, nutrients such as glucose have been shown to repress proteolytic activity in yeasts, Bacilli, Escherichia coli and other microorganisms.
Extracellular proteases usually have wide substrate specificities and can degrade most non-structural proteins, such as albumin, casein, insulin or hemoglobin. For example, many marine bacteria produce extracellular proteases in order to utilize proteinaceous macromolecules which accumulate on surfaces in the marine environment. Many pathogenic microorganisms secrete proteases, some of which are involved in the infection process. Several microbial species release specific proteases, including the collagenases, the elastases and the keratinases, which can hydrolyze structural and connective tissue proteins which are resistant to attack by most proteases.
Proteases are classified by their catalytic mechanism into four groups. These include: 1) serine proteases, 2) cysteine proteases, 3) aspartic proteases and 4) metallo-proteases. Characteristically, the enzymes of the metalloprotease category have an optimal pH between 5 and 9 and are sensitive to metal-chelating reagents, such as ethylenediaminetetra-acetic acid (EDTA), but are unaffected by serine proteinase inhibitors or sulfhydryl agents. However, many of the EDTA-inhibited enzymes can be reactivated by the addition of ions, such as zinc, calcium, or cobalt.
Metalloproteases are widespread, although only a few have been reported in fungi. Most metalloproteases can be divided into three groups: acid, neutral or alkaline proteases. Characteristically, bacterial metalloproteases are zinc-containing enzymes, with one atom of zinc per molecule of enzyme. The zinc atom is essential for enzyme activity.
Moreover, calcium ions have been shown to stabilize the protein structure of the zinc-containing metalloproteases. However, the amount of calcium in a metalloprotease varies from four atoms per molecule for the Bacillus thermoproteo-lyticus thermolysin to less than 0.2 atoms per molecule for the Aeromonas proteolytica enzyme. The four calcium ions associated with thermolysin permit the enzyme to withstand increased temperature via a cooperative mechanism, while removal of Ca.sup.2+ ions from the native enzyme results in an irreversible loss of catalytic activity at temperatures below 50.degree. C., and irreversible structural changes at temperatures above 50.degree. C.
In contrast, although the exoprotease produced by Bacillus amyloliquefaciens has properties similar to thermolysin, the enzyme molecule contains only two calcium ions and is considerably less heat stable. Thus, the ability of an alkaline metalloprotease to withstand high temperatures appears to be a function of the calcium ions associated with the enzyme.
The thermostable proteases currently in industrial use belong primarily to either the neutral metalloprotease or the alkaline serine groups. The neutral metalloproteases include thermolysin, which is stable up to 80.degree. C. Proteases of this type are exemplified by cultures of Thermus aquaticus, such as taught by U.S. Pat. Nos. 4,889,818 and 4,442,214. However, neutral metalloproteases are inactivated by alkaline conditions, and have poor stability to oxidizing agents.
Alkaline serine proteases include the subtilisins, which have an optimal temperature of 60.degree. C. Most commercially available alkaline proteases are produced by microorganisms belonging to the Bacillus and Aspergillus genera, although some are produced by Streptomyces, Arthrobacter, and Fusarium. Enzymes produced by cultivation of the genus Bacillus constitute the majority of proteolytic enzymes in present use, such as those described in U.S. Pat. Nos. 4,797,362, 4,771,003, and 3,871,963. Further, numerous other Bacillus-produced proteases are used in detergent washing compositions, such as those described in U.S. Pat. Nos. 4,764,470, 4,052,262, 4,002,572, and 3,827,938.
Bacillus alkaline proteases have broad specificity and certain species exhibit activity at elevated levels of alkalinity. Disadvantageously however, Bacillus alkaline proteases have less than desirable stability to oxidizing agents, such as hypochlorite or hydrogen peroxide, sensitivity to diisopropylphosphofluoridate and phenylmethylsulfonyl fluoride, and are completely unstable in chlorine bleaches. Furthermore, Bacillus culture results in the production of heat-resistant endospores, antibiotics and undesirable enzymes, in addition to the production of proteases.
In contrast, members of the Aspergillus species are non-pathogenic and non-toxic. However, mutants of the organisms are rather unstable. Thus, strain improvement is essentially rendered unfeasible.
Certain other bacteria are known to produce alkaline exoproteases which are characterized by stability over limited pH and temperature ranges, although each such protease is readily distinguished physically and biochemically from the protease produced by the present process. Alkaline proteases include those which are produced, for example, in accordance with U.S. Pat. Nos. 4,965,197, 4,865,983, and 4,390,629.
It is evident that there is a long-felt need in the art for thermostable proteolytic enzymes in industry and medicine. For example, until the present invention, the art has been unable to fulfill the need for thermostable alkaline exoproteases which can be used in preparing laundry detergents or in formulating animal feed, as catalysts in the drug and chemical industry, as reagents for converting starch to sugars, for destroying waste products or converting waste protein to useful alternative chemicals, for tanning, for generating peptides from cloned precursors, and in the dissolution of necrotic tissue or blood clots, and the like. Additional uses of proteolytic enzymes will undoubtedly be found.
In view of the foregoing, it is evident that although numerous proteases are known, that the prior art has not produced proteases which completely satisfy the need in the art for reliable, thermostable alkaline exoproteases. Accordingly, the identification and characterization of at least one novel thermostable alkaline exoprotease which is highly active on proteinaceous material at both high pH and elevated temperatures, as well as related methods of preparation and use, would significantly advance the art. The present invention satisfies this need and provides related advantages in the art.